Processed ecm materials with enhanced component profiles

ABSTRACT

Described are medical graft materials and devices having improved properties relating to their component profiles.

REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/178,321, filed Jul. 23, 2008, pending which is a continuation ofInternational Application No. PCT/US2007/082238 filed Oct. 23, 2007,entitled “PROCESSED ECM MATERIALS WITH ENHANCED COMPONENT PROFILES”which claims the benefit of U.S. Provisional Patent Application Ser. No.60/853,584 filed Oct. 23, 2006, entitled “PROCESSED ECM MATERIALS WITHENHANCED COMPONENT PROFILES” both of which are hereby incorporated byreference in their entirety.

BACKGROUND

The present invention relates generally to medical graft materials and,in particular, to medical graft materials derived from tissue materials.

A variety of processed extracellular matrix (ECM) materials have beenproposed for use in medical grafting, cell culture, and other relatedapplications. For instance, medical grafts and cell culture materialscontaining submucosa derived from small intestine, stomach or urinarybladder tissues, have been proposed. See, e.g., U.S. Pat. Nos.4,902,508, 4,956,178, 5,281,422, 5,554,389, 6,099,567 and 6,206,931. Inaddition, Cook Biotech Incorporated, West Lafayette, Ind., currentlymanufactures a variety of medical products based upon small intestinalsubmucosa under the trademarks SURGISIS®, STRATASIS® and OASIS®.

Medical materials derived from liver basement membrane have also beenproposed, for example in U.S. Pat. No. 6,379,710. As well, ECM materialsderived from amnion (see e.g. U.S. Pat. Nos. 4,361,552 and 6,576,618)and from renal capsule membrane (see International PCT PatentApplication No. WO 03/002165 published Jan. 9, 2003) have been proposedfor medical and/or cell culture applications.

Attempts have been made to provide a processed ECM material that retainsmedically significant substances other than collagen. However, in orderto prepare a processed ECM in which undesired components have beenremoved, the material is typically subjected to a battery ofmanipulations, which can have undesirable consequences to the desirablecomponents contained within the material. For example, submucosa andother ECM materials have been shown to include a variety of desirablecomponents other than collagen that can contribute to the bioactivity ofthe materials and to their value in medical grafting and other uses. Asexamples, ECM materials can include growth factors, cell adhesionproteins, and proteoglycans that can be beneficial when retained in theprocessed ECM. However, it is difficult to selectively retain thesecomponents while removing high levels of undesired components inpreparing a medically acceptable graft.

Needs remain for biomaterials that not only possess the necessaryphysical properties and high levels of biocompatibility and sterility,but also the desired levels of beneficial components. Methods forpreparing and using these materials, as well as medical devices formedfrom these materials are also needed. The present invention addressesthese needs.

SUMMARY

In one aspect, the present invention provides a medical graft materialincluding a processed extracellular matrix (ECM) material. The ECMmaterial retains collagen and non-collagen components, and desirablyexhibits an angiogenic character. At the same time, the ECM material haslow levels of undesired components such as native lipids, nucleic acids(e.g. DNA), and/or immunoglobulin A (IgA) components. In preferredembodiments, the ECM material includes submucosa.

In one embodiment, the present invention provides a medical graftmaterial including a sterile, decellularized extracellular matrix (ECM)material. The ECM material includes native fibroblast growth factor-2(FGF-2), and native immunoglobulin A (IgA) at a level of no greater than20 μg/g. In some forms, the material can have a lipid content of nogreater than about 4%. In preferred embodiments, the isolated ECMmaterial includes submucosa and has a native IgA at a level of nogreater than 5 μg/g and a native lipid content of no greater than about3%.

In yet another aspect, the present invention provides a medical graftmaterial including a sterile, decellularized extracellular matrix (ECM)material. The ECM material has a native FGF-2 content of at least about10 ng/g and at least one of, and in certain forms each of (i) native IgAat a level of no greater than about 20 μg/g; (ii) native lipids at alevel of no greater than about 4% by weight; (iii) native hyaluronicacid at a level of at least about 50 μg/g; and (iv) native sulfatedglycosaminoglycan at a level of at least about 500 μg/g. In certainpreferred embodiments, the ECM material includes submucosa. Inadditional forms, the ECM material has a native IgA at a level of nogreater than about 5 μg/g and a native lipid content of no greater thanabout 3% by weight.

Further provided by the invention is a method for treating a patient.The method includes grafting the patient with a medical graft materialof the invention.

The present invention further provides a method for preparing a medicalgraft material. The method comprises providing a starting extracellularmatrix (ECM) material. The starting ECM material is treated to decreasethe lipid, nucleic acid and/or IgA and/or other immunoglobulin contentof the material while the material retains a significant level of growthfactor(s), proteoglycans, and/or glycosaminoglycans. In certainembodiments, the method includes treating the starting ECM material witha dilute ionic detergent solution to disrupt cell and nuclear membranes,and with a basic solution to solubilize and remove DNA and other nucleicacid materials. The method can additionally include treating the ECMmaterial with an organic solvent to remove lipids from the material,and/or with an oxidizing disinfectant solution, e.g. containing a peroxycompound, to disinfect the material. The ECM material is preferablyrinsed to remove residues left by these solutions.

Medical graft materials of the invention can be provided in a widevariety of forms. For example, a medical graft material can be providedas one or more sheets, a paste, a sponge, a non-gelled aqueous solution,a powder, or a gel. Combinations of these forms are also contemplated.

The various forms of a medical graft material can be used in a widevariety of medical (including veterinary) applications. Examples includethe repair or reconstruction of tissue, such as nervous tissue, dermaltissue (e.g. in wound care), cardiovascular tissue (including vasculartissue and cardiac tissue), pericardial tissue, muscle tissue, bladdertissue, ocular tissue, periodontal tissue, bone, connective tissue suchas tendons or ligaments, and others.

Additional embodiments as well as features and advantages of theinvention will be apparent from the descriptions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a medical graft material of the invention formed from asingle layer of an extracellular matrix (ECM) material isolated fromtissue of a warm blooded vertebrate.

FIG. 2 depicts a medical graft material of the invention formed from twolayers of an ECM material isolated from tissue of a warm bloodedvertebrate.

FIG. 3 provides a side view of one prosthetic valve device of theinvention that includes an ECM material attached to a frame.

FIG. 4 provides a left side view of the prosthetic valve device depictedin FIG. 3.

FIG. 5 provides a right side view of the prosthetic valve devicedepicted in FIG. 3.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments thereof andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications in the described embodiments, and such furtherapplications of the principles of the invention as illustrated hereinbeing contemplated as would normally occur to one skilled in the art towhich the invention relates.

As disclosed above, in one aspect the invention provides extracellularmatrix graft materials having unique component profiles that are low inundesired components while retaining significant levels of desiredcomponents. These unique materials can be prepared by processing methodsthat comprise treating a relatively impure ECM starting material todecrease the content of the undesired components, such as nucleic acid,lipids and/or immunoglobulins such as IgA, while retaining substantiallevels of desired components such as growth factor(s), proteoglycansand/or glycosaminoglycans (GAGs). Typically, the ECM starting materialwill be treated with a mild detergent solution, such as an ionic ornonionic detergent solution. The low concentration of detergent enablesa retention of a substantial level of desired components, such as thoseas noted above. In certain modes of operation, the ECM material will betreated with an aqueous solution of sodium dodecyl sulfate (SDS) oranother ionic or nonionic detergent at a detergent concentration ofabout 0.05% to about 1%, more preferably about 0.05% to about 0.3%. Thistreatment can be for a period of time effective to disrupt cell andnuclear membranes and to reduce the immunoglobulin (e.g. IgA) content ofthe ECM material, typically in the range of about 0.1 hour to about 10hours, more typically in the range of about 0.5 hours to about 2 hours.Processing the isolated ECM material in this manner preferably disruptscell and nuclear membranes and results in a material with asubstantially reduced its IgA content, thus reducing the immunogenicityof the material. For example, a processed ECM material of the inventioncan have a native IgA content of no greater than about 20 μg/g. Inpreferred embodiments, an ECM material of the invention can have anative IgA content of no greater than 15 μg/g, no greater than 10 μg/g,or even no greater than 5 μg/g. In certain embodiments, the processedECM material includes essentially no native IgA. By “essentially no IgA”is meant that the isolated ECM material includes IgA below detectablelevels. Means for detecting IgA are well known in the art and include,for example, enzyme-linked immunosorbent assay (ELISA). It will beunderstood in this regard that ECM materials obtained from differentsources may have differing immunoglobulins that predominate in thetissue. It is expected that the processing techniques disclosed hereinwill be effective to reduce the content of ECM materials in otherimmunoglobulins, including those that predominate in the source tissue.Accordingly, other aspects of the invention relate to the isolation ofan ECM material that has substantially reduced levels (e.g. less thanabout 20 μg/g) of (i) the predominant immunoglobulin in the sourcetissue, or (ii) the total immunoglobulin content (the sum of allimmunoglobulins in the tissue).

In addition to treating an ECM material with a detergent medium, the ECMmaterial can be contacted with other agents that participate inachieving the desired ECM component profile. For example, the ECMmaterial can be treated with an aqueous medium, preferably basic, inwhich DNA is soluble. Such a medium can in certain forms have a pH inthe range of above 7 to about 9, with pH's in the range of about 8 toabout 8.5 proving particularly beneficial in some embodiments. The basicaqueous medium can include a buffer, desirably a biocompatible buffersuch as tris(hydroxymethyl)aminomethane (TRIS), and/or a chelating agentsuch as ethylene diamine tetraacetic acid (EDTA). In one preferred form,the nucleic acid solubilizing medium is a TRIS-borate-EDTA (TBE) buffersolution. In another preferred form, the nucleic acid solubilizingmedium is a solution of ammonium hydroxide. This treatment with a DNAsolubilizing medium can be for a period of time effective to reduce theDNA content of the ECM material, typically in the range of about 0.1hour to about 10 hours, more typically in the range of about 0.5 hoursto about 2 hours.

In addition to treatment with detergent and DNA-solubilization media,methods of preparing medical graft materials of the invention caninvolve treatment with a liquid medium that results in a substantialreduction of the level of lipid components of the ECM material. Forexample, the resulting native lipid content of the ECM material can bereduced to no greater than about 4% in certain embodiments. This can beaccomplished, for example, by a preparative process that involves a stepof treating the ECM material with a liquid organic solvent in which thelipids are soluble. Suitable such organic solvents include for examplewater-miscible solvents, including polar organic solvents. These includelow molecular weight (e.g. C₁ to C₄) alcohols, e.g. methanol, ethanol,isopropanol, and butanols, acetone, chloroform, and others. Additionalorganic solvents include nonpolar solvents such as hexane, benzene,toluene and the like. In more preferred embodiments, the processed ECMmaterial will be processed to have a native lipid content no greaterthan about 3%, or no greater than about 2.5%. This treatment with alipid-removing medium can be for a period of time effective to reducethe lipid content of the ECM material, typically in the range of about0.1 hour to about 10 hours, more typically in the range of about 0.1hours to about 1 hours. In certain embodiments, multiple (two or more)such treatments will be conducted. Additionally, treatment with thelipid-reducing medium as discussed above can be carried out before orafter treatment with a detergent medium and/or aqueous (preferablybasic) DNA-reducing medium as discussed above. In certain preferredembodiments, treatment with the lipid-reducing medium will occur beforetreatment with the detergent medium and/or the aqueous (preferablybasic) medium.

The ECM material can also be treated with a disinfecting solution. Thedisinfecting solution can include a disinfecting agent such as analcohol, a peroxy compound, or another oxidizing or non-oxidizingdisinfectant. In certain forms, the disinfecting solution will be aperacetic acid solution having a peracetic acid concentration of about0.1% to about 0.3%. Peracetic acid and other oxidizing disinfectanttreatment solutions such as those described in U.S. Pat. No. 6,206,931can be used, for example.

The ECM material also can be rinsed at various stages throughout itspreparation (e.g., with tap water, high purity water or buffer) so as toremove introduced chemical residues that remain in or on the material.In preferred embodiments, at least about 90% of detergent residues areremoved from the material. More preferably, at least about 95%, or atleast about 97% of detergent residues are removed from the material.

Treatments with detergent, DNA-solubilizing, lipid-solubilizing,rinsing, and potentially other liquid media can be carried out in anysuitable fashion and in any suitable order wherein the ECM material iscontacted with the medium. For example, the ECM material can be soakedin the medium, potentially with agitation, for the duration of thetreatment. Other contacting methods such as spraying or showering theECM material with the medium can also be used. As well, the treatment(s)can be carried out at any suitable temperature. Temperatures of 0° C. toabout 50° C. are preferred as they enable minimizing or avoidingsubstantial denaturing of the collagen and other desirable components ofthe ECM material. More preferably, the treatment temperature will be inthe range of about 0° C. to about 37° C., and more typically in therange of about 20° C. to about 37° C. It will be understood, however,that other temperatures may be used within the broader aspects of theinvention. In embodiments where a tubular or other closable structure isformed, the ECM material including a lumen can be clamped at one end toallow the lumen to be filled with medium and can be clamped at the otherend to essentially close a proximal and distal end of the tubular ECMmaterial. The tubular ECM material having a filled lumen can besubmerged in a medium, which can be the same or different medium. Inthis way, each of the lumen and the outer surface of a tubular ECMmaterial can be treated without necessarily requiring that the treatmentmedium diffuses or otherwise passes through the ECM material. Thisprocess can be repeated with any medium used herein, including anyrinsing step.

Treatments such as those above, and/or other chemical and/or mechanicaltreatments, will decellularize the ECM tissue, desirably resulting in aprocessed ECM tissue that is free of viable cells derived from thesource tissue. Acellular ECM material so obtained can, however, be usedto culture or be seeded with cells in certain embodiments, for examplecertain of those described below.

Processed ECM materials of the invention can be derived from anysuitable organ or other tissue source, desirably one containingsignificant collagenous connective tissue. Human or other animal tissuesources can be used. Non-human animal sources can be warm-bloodedvertebrates, including mammals, with bovine, ovine, caprine, and porcinesources being suitable. Suitable ECM materials obtained from thesetissue sources can include submucosa, renal capsule membrane, dermalcollagen, dura mater, pericardium, fascia lata, serosa, peritoneum orbasement membrane layers, including liver basement membrane. Suitablesubmucosa materials for these purposes include, for instance, intestinalsubmucosa, including small intestinal submucosa, stomach submucosa,urinary bladder submucosa, and uterine submucosa. It will be wellunderstood that in isolating ECMs that include submucosa, some or all ofthe original submucosa from the source tissue may be retained,potentially along with materials derived from one or more adjacenttissue layers. Similar principles apply to other collagen-rich layers orother tissues named herein—the recovered ECM material may include someor all of the specified tissue originally present in the source tissue,and/or may remain connected to adjacent tissue(s) in the final processedECM material.

Processed, naturally-derived ECM materials of the invention willtypically include abundant collagen, most commonly being constituted atleast about 80% by weight collagen on a dry weight basis. Suchnaturally-derived ECM materials will for the most part include collagenfibers that are non-randomly oriented, for instance occurring asgenerally uniaxial or multi-axial but regularly oriented fibers. Whenprocessed to retain native bioactive components, the ECM material canretain these components interspersed as solids between, upon and/orwithin the collagen fibers. Particularly desirable naturally-derived ECMmaterials for use in the invention will include significant amounts ofsuch interspersed, non-collagenous solids that are readily ascertainableunder light microscopic examination. Such non-collagenous solids canconstitute a significant percentage of the dry weight of the ECMmaterial in certain inventive embodiments, for example at least about1%, at least about 3%, and at least about 5% by weight in variousembodiments of the invention.

The processed ECM material of the present invention may also exhibit anangiogenic character and thus be effective to induce angiogenesis in ahost engrafted with the material. In this regard, angiogenesis is theprocess through which the body makes new blood vessels to generateincreased blood supply to tissues. Thus, angiogenic materials, whencontacted with host tissues, promote or encourage the formation of newblood vessels. Methods for measuring in vivo angiogenesis in response tobiomaterial implantation have been developed. For example, one suchmethod uses a subcutaneous implant model to determine the angiogeniccharacter of a material. See, C. Heeschen et al., Nature Medicine 7(2001), No. 7, 833-839. When combined with a fluorescencemicroangiography technique, this model can provide both quantitative andqualitative measures of angiogenesis into biomaterials. C. Johnson etal., Circulation Research 94 (2004), No. 2, 262-268.

It is advantageous to prepare bioremodelable ECM materials for themedical graft materials and methods of the present invention. Suchmaterials that are bioremodelable and promote cellular invasion andingrowth provide particular advantage. Bioremodelable materials may beused in this context to promote cellular growth within the site in whicha medical graft material of the invention is implanted.

As noted above, the processed submucosal (submucosa-containing) ECMmaterial and any other ECM material may retain any of a variety ofgrowth factors or other beneficial bioactive components native to thesource tissue. For example, the submucosa or other ECM can include oneor more native growth factors such as basic fibroblast growth factor(FGF-2), transforming growth factor beta (TGF-beta), epidermal growthfactor (EGF), connective tissue growth factor (CTGF), vascularendothelial growth factor (VEGF) and/or platelet derived growth factor(PDGF). As well, submucosa or other ECM used in the invention mayinclude other biological materials such as proteoglycans and/orglycosaminoglycans, such as heparin, heparin sulfate, hyaluronic acid,fibronectin and the like. Thus, generally speaking, the processed ECMmaterial will include at least one native bioactive component thatinduces, directly or indirectly, a cellular response such as a change incell morphology, proliferation, growth, protein or gene expression.

In preferred embodiments, the processed ECM material will exhibit acomponent profile wherein the following non-collagen components arepresent in the stated amounts:

Component Preferred Range More Preferred Range Lipid: less than 5% lessthan 3% FGF-2: greater than 2 ng/g greater than 5 ng/g IgA: less than 5μg/g less than 1 μg/g HA: greater than 50 μg/g greater than 100 μg/gsGAG: greater than 1000 μg/g greater than 2000 μg/g Visible nuclei lessthan 200 per less than 100 per 0.263 mm² 0.263 mm²

Further, in addition to the retention of native bioactive components,non-native bioactive components such as those synthetically produced byrecombinant technology or other methods, may be incorporated into thesubmucosal or other ECM material. These non-native bioactive componentsmay be naturally-derived or recombinantly produced proteins thatcorrespond to those natively occurring in the ECM tissue, but perhaps ofa different species (e.g. human proteins applied to collagenous ECMsfrom other animals, such as pigs). The non-native bioactive componentsmay also be drug substances. Illustrative drug substances that may beincorporated into and/or onto the ECM materials used in the inventioninclude, for example, antibiotics, thrombus-promoting substances such asblood clotting factors, e.g. thrombin, fibrinogen, and the like. Thesesubstances may be applied to the ECM material as a premanufactured step,immediately prior to the procedure (e.g. by soaking the material in asolution containing a suitable antibiotic such as cefazolin), or duringor after engraftment of the material in the patient.

A non-native bioactive component can be applied to a submucosa or otherECM tissue by any suitable means. Suitable means include, for example,spraying, impregnating, dipping, etc. The non-native bioactive componentcan be applied to the ECM tissue either before or after the material isaffixed to an elongate member. Similarly, if other chemical orbiological components are included in the ECM tissue, the non-nativebioactive component can be applied either before, in conjunction with,or after these other components.

Processed submucosal or other ECM tissue of the invention preferablyexhibits an endotoxin level of less than about 12 endotoxin units (EU)per gram, more preferably less than about 5 EU per gram, and mostpreferably less than about 1 EU per gram. As additional preferences, thesubmucosa or other ECM material may have a bioburden of less than about1 colony forming units (CFU) per gram, more preferably less than about0.5 CFU per gram. Fungus levels are desirably similarly low, for exampleless than about 1 CFU per gram, more preferably less than about 0.5 CFUper gram. Nucleic acid levels are preferably less than about 2 μg/mg,more preferably less than about 1 μg/mg, and virus levels are preferablyless than about 50 plaque forming units (PFU) per gram, more preferablyless than about 5 PFU per gram.

In certain embodiments, endotoxin levels can be considered in relationto the surface area of one or more isolated, single sheets of an ECMmaterial. In such instances, a sheet of ECM material can exhibit anendotoxin level of less than about 0.25 EU/cm². In preferredembodiments, a sheet of ECM material exhibits an endotoxin level of lessthan about 0.2 EU/cm², less than about 0.1 EU/cm², and even less thanabout 0.05/cm². In a most preferred embodiment, a sheet of ECM materialexhibits an endotoxin level of less than about 0.025 EU/cm². MultilayerECM structures including a plurality of bonded or otherwise coupledsheets of ECM material can exhibit similar endotoxin levels based on thesurface area of the overall multilayer structure.

The processed ECM material of the invention can be packaged or otherwisestored in a dehydrated or hydrated state. Dehydration of a medical graftmaterial of the invention can be achieved by any means known in the art.Preferably, dehydration is accomplished by either lyophilization orvacuum pressing, although other techniques, for example air drying, canalso be used. When stored in a dry state, it will often be desirable torehydrate the processed ECM material prior to use. In this regard, anysuitable wetting medium can be used to rehydrate the medical material,including as examples water or buffered saline solutions.

In certain embodiments, the processed ECM material can be crosslinked.Increasing the amount (or number) of crosslinkages within the medicalgraft material or between two or more layers of the medical graftmaterial can be used to enhance its strength. However, crosslinkageswithin the medical graft material may also effect its bioremodelabilityor other bioactive characteristics. Consequently, in certainembodiments, a bioremodelable ECM material will be provided thatsubstantially retains its native level of crosslinking, or the amountand/or type of added crosslinks within the ECM material can bejudiciously selected to retain the desired level of bioremodelability orother bioactive characteristic.

For use in the present invention, any introduced crosslinking of theprocessed ECM material may be achieved by photo-crosslinking techniques,or by the application of a crosslinking agent, such as by chemicalcrosslinkers, or by protein crosslinking induced by dehydration or othermeans. Chemical crosslinkers that may be used include for examplealdehydes such as glutaraldehydes, diimides such as carbodiimides, e.g.,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, ribose orother sugars, acyl-azide, sulfo-N-hydroxysuccinamide, or polyepoxidecompounds, including for example polyglycidyl ethers such asethyleneglycol diglycidyl ether, available under the trade name DENACOLEX810 from Nagese Chemical Co., Osaka, Japan, and glycerol polyglycerolether available under the trade name DENACOL EX 313 also from NageseChemical Co. Typically, when used, polyglycerol ethers or otherpolyepoxide compounds will have from 2 to about 10 epoxide groups permolecule. Preferably, a medical graft material is crosslinked with acrosslinking agent comprising transglutaminase.

Processed ECM materials of the invention can be manufactured into avariety of physical forms to suit a variety of medical applications. Forexample, an isolated ECM material can be provided as one or more sheets,a paste, a foam, a non-gelled aqueous solution, a powder, or a gel.Combinations of these forms are also contemplated. In this regard, theconfiguration of the ECM material may be attained before or after theECM material has been processed as described herein. Further, an ECMcomposite material can be manufactured in larger, bulk dimensions, andthen divided into smaller products. Moreover, the ECM material mayprovided in a naturally-derived layer form, or may itself be amanufactured article, such as a sponge or cast sheet, prepared from anaturally-derived ECM material.

Medical graft materials of the invention may be used in a wide varietyof medical (including veterinary) applications. Examples include therepair or reconstruction of tissue, such as nervous tissue, dermaltissue such as in wound healing, e.g. application to external dermalwounds, including but not limited to ulcers (e.g. diabetic or otherchronic ulcers), cardiovascular tissue (including vascular tissue andcardiac tissue), pericardial tissue, muscle tissue, ocular tissue,periodontal tissue, bone, connective tissue such as tendons orligaments, in the treatment of gastrointestinal fistulae (e.g. processedinto the form of a plug to occlude at least the primary opening of afistula such as an anorectal, rectovaginal, or enterocutaneous fistula),and others.

In one embodiment, the processed ECM material is made into a fluidizedcomposition, for instance using techniques as described in U.S. Pat.Nos. 5,275,826 and 5,516,533. In this regard, solutions or suspensionsof the ECM material can be prepared by comminuting and/or digesting thematerial with a protease (e.g. trypsin or pepsin), for a period of timesufficient to solubilize the material and form substantially homogeneoussolution. The ECM material is desirably comminuted by tearing, cutting,grinding, shearing or the like. Grinding the material in a frozen orfreeze-dried state is advantageous, although good results can beobtained as well by subjecting a suspension of pieces of the material totreatment in a high speed blender and dewatering, if necessary, bycentrifuging and decanting excess waste. The comminuted material can bedried, for example freeze dried, to form a powder. Thereafter, ifdesired, the powder can be hydrated, that is, combined with water orbuffered saline and optionally other pharmaceutically acceptableexcipients, to form a fluid tissue graft composition, e.g. having aviscosity of about 2 to about 300,000 cps at 25° C. The higher viscositygraft compositions can have a gel or paste consistency.

A fluidized ECM material of this invention finds use as an injectableheterograft for tissues, for example, bone or soft tissues, in need ofrepair or augmentation most typically to correct trauma ordisease-induced tissue defects. The present fluidized compositions arealso used advantageously as a filler for implant constructs comprising,for example, one or more sheets of a collagenous ECM material formedinto sealed (sutured) pouches for use in cosmetic or trauma-treatingsurgical procedures.

In one illustrative preparation, an ECM material prepared as describedherein is reduced to small pieces (e.g. by cutting) which are charged toa flat bottom stainless steel container. Liquid nitrogen is introducedinto the container to freeze the specimens, which are then comminutedwhile in the frozen state to form a coarse powder. Such processing canbe carried out, for example, with a manual arbor press with acylindrical brass ingot placed on top of the frozen specimens. The ingotserves as an interface between the specimens and the arbor of the press.Liquid nitrogen can be added periodically to the specimens to keep themfrozen.

Other methods for comminuting ECM material specimens can be utilized toproduce a powder usable in accordance with the present invention. Forexample, ECM material specimens can be freeze-dried and then groundusing a manual arbor press or other grinding means. Alternatively, ECMmaterial can be processed in a high shear blender to produce, upondewatering and drying, a powder.

Further grinding of the ECM material powder using a prechilled mortarand pestle can be used to produce consistent, more finely dividedproduct. Again, liquid nitrogen is used as needed to maintain solidfrozen particles during final grinding. The powder can be easilyhydrated using, for example, buffered saline to produce a fluidizedtissue graft material of this invention at the desired viscosity.

To prepare another preferred fluidized material, an ECM material powdercan be sifted through a wire mesh, collected, and subjected toproteolytic digestion to form a substantially homogeneous solution. Forexample, the powder can be digested with 1 mg/ml of pepsin (SigmaChemical Co., St. Louis Mo.) and 0.1 M acetic acid, adjusted to pH 2.5with HCl, over a 48 hour period at room temperature. After thistreatment, the reaction medium can be neutralized with sodium hydroxide(NaOH) to inactivate the peptic activity. The solubilized submucosa canthen be concentrated by salt precipitation of the solution and separatedfor further purification and/or freeze drying to form aprotease-solubilized collagenous ECM material in powder form.

Fluidized compositions of this invention find wide application in tissuereplacement, augmentation, and/or repair. The fluidized compositions canbe used to induce regrowth of natural connective tissue or bone in anarea of an existent defect. By injecting an effective amount of afluidized composition into the locale of a tissue defect or a wound inneed of healing, one can readily take advantage of the biotropicproperties of the collagenous ECM material.

In orthopedic applications, a medical graft material of the inventioncan be used to repair bone tissue, for instance using the generaltechniques described in U.S. Pat. No. 5,641,518. Thus, a powder form ofthe material can be implanted into a damaged or diseased bone region forrepair. The powder can be used alone, or in combination with one or moreadditional bioactive agents such as physiologically compatible minerals,growth factors, antibiotics, chemotherapeutic agents, antigen,antibodies, enzymes and hormones. Preferably, the powder-form implantwill be compressed into a predetermined, three-dimensional shape, whichwill be implanted into the bone region and will substantially retain itsshape during replacement of the graft with endogenous tissues.

A processed ECM material of the invention can also be used as a cellgrowth substrate, illustratively in sheet, paste or gel form incombination with nutrients which support the growth of the subjectcells, e.g. eukaryotic cells such as endothelial, fibroblastic, fetalskin, osteosarcoma, and adenocarcinoma cells (see, e.g. InternationalPCT Application Publication No. WO 96/24661). In preferred forms, thesubstrate composition will support the proliferation and/ordifferentiation of mammalian cells, including human cells.

A processed ECM material of the invention can also be used in body wallrepair, including for example in the repair of abdominal wall defectssuch as hernias, using techniques analogous to those described in Ann.Plast. Surg., 1995, 35:374-380; and J. Surg. Res., 1996, 60:107-114. Insuch applications, preferred medical graft materials of the inventionpromote favorable organization, vascularity and consistency in theremodeled tissue. In dermatological applications, a medical graftmaterial of the invention can be used in the repair of partial or fullthickness wounds and in dermal augmentation using general graftingtechniques which are known to the art and literature (see, e.g. Annalsof Plastic Surgery 1995, 35:381-388). In addition, in the area of burntreatment, it is generally known to provide a dermal substitute ontowhich cultured epidermal grafts (preferably cultured epidermalautografts, or CEA's) are transplanted. Such cultured grafts havetypically involved transplanting keratinocytes and/or fibroblasts ontothe dermal substitute. In accordance with the present invention, themedical graft material can be used as the dermal substitute, for examplein sheet form, and the CEA accordingly transplanted onto the material.In one mode of practicing this aspect of the invention, keratinocytescan be transplanted, for example by seeding or transferring akeratinocyte sheet, onto the mucosal side of the submucosa. Fibroblastscan be transplanted also on the mucosal and/or on the opposite(abluminal) side of the submucosa.

The processed ECM material of the invention can also be used in tissuegrafting in urogenital applications. For instance, the medical graftmaterial can be used in urinary bladder repair to provide a scaffold forbladder regeneration, using techniques corresponding to those generallydescribed in U.S. Pat. No. 5,645,860; Urology, 1995, 46:396-400; and J.Urology, 1996, 155:2098. In fluidized form, the inventive medical graftmaterial can also find use in an endoscopic injection procedure tocorrect vesicureteral reflux. In such applications, an injection can bemade, for instance in the area under the ureteral orifice of a patient,to induce smooth muscle growth and collagen formation at the injectionsite.

Generally, when configured for use as a tissue graft, the processed ECMmaterial of the invention can include one or more sheets of ECM materialthat can be cut or otherwise configured to a desired size for its enduse. The graft material is in many instances sized larger than thetissue defect to which it is applied. Sizing the medical graft materialin this way allows for easy attachment to the surrounding tissue.

Once the sized ECM graft material has been placed on, in, or around thedefect, the material can be attached to the surrounding tissue using anyof several known suitable attachment means. Suitable attachment meansinclude, for example, biocompatible adhesives (e.g., fibrin glue),stapling, suturing, and the like. Preferably, the medical graft materialis attached to the surrounding tissue by sutures. There are a variety ofsynthetic materials currently available in the art for use as sutures.For example, sutures comprising Prolene™, Vicryl™, Mersilene™,Panacryl™, and Monocryl™, are contemplated for use in the invention.Other suture materials will be well known to those skilled in the art.The aforementioned materials therefore serve merely as examples and,consequently, are in no way limiting.

In other areas, medical graft materials formed with an ECM material ofthe present invention can be used in neurologic applications, forexample in techniques requiring a dural substitute to repair defects dueto trauma, tumor resection, or decompressive procedures.

In sheet form, a processed ECM medical graft material of the inventioncan be comprised of a single layer or multiple layers of material. Thus,in certain embodiments, a single isolated layer of ECM material or amultilaminate ECM construct can be used. Illustrative multilaminate ECMconstructs for use in the invention may, for example, have from two toabout ten isolated ECM layers laminated together.

Multilaminate ECM constructs for use in the invention can be prepared inany suitable fashion. In this regard, a variety of techniques forlaminating ECM layers together can be used. These include, for instance,dehydrothermal bonding under heated, non-heated or lyophilizationconditions, using adhesives, glues or other bonding agents, crosslinkingwith chemical agents or radiation (including UV radiation), or anycombination of these with each other or other suitable methods. Foradditional information as to multilaminate ECM constructs that can beused in the invention, and methods for their preparation, reference maybe made for example to U.S. Pat. Nos. 5,711,969, 5,755,791, 5,855,619,5,955,110, 5,968,096, and to U.S. Patent Application Publication No.20050049638.

Single layer ECM or multilaminate ECM constructs or other biocompatiblematerials used in the present invention can have or can lackperforations or slits in their structure, and in certain embodiments canhave a meshed structure for example as described in U.S. ApplicationPatent Publication No. 20050021141. Such mesh patterned structures canbe used to provide an ECM or other implant segment that is highlydeformable for use in the present invention.

In additional embodiments, processed ECM's of the invention can besubjected to processes that expand the materials. In certain forms, suchexpanded materials can be formed by the controlled contact of an ECMmaterial with one or more alkaline substances until the materialexpands, and the isolation of the expanded material. Illustratively, thecontacting can be sufficient to expand the ECM material to at least 120%of (i.e. 1.2 times) its original bulk volume, or in some forms to atleast about two times its original volume. Thereafter, the expandedmaterial can optionally be isolated from the alkaline medium, e.g. byneutralization and/or rinsing. The collected, expanded material can beused in any suitable manner in the preparation of a medical device.Illustratively, the expanded material can be enriched with bioactivecomponents, dried, and/or molded, etc., in the formation of a graftconstruct of a desired shape or configuration. In certain embodiments, amedical graft material and/or device formed with the expanded ECMmaterial can be highly compressible (or expandable) such that thematerial can be compressed for delivery, such as from within the lumenof a cannulated delivery device, and thereafter expand upon deploymentfrom the device so as to become anchored within a patient and/or causeclosure of a tract within the patient.

Expanded ECM materials can be formed by the controlled contact of aprocessed ECM material as described above with an aqueous solution orother medium containing sodium hydroxide. Alkaline treatment of thematerial can cause changes in the physical structure of the materialthat in turn cause it to expand. Such changes may include denaturationof the collagen in the material. In certain embodiments, it is preferredto expand the material to at least about three, at least about four, atleast about 5, or at least about 6 or even more times its original bulkvolume. The magnitude of the expansion is related to several factors,including for instance the concentration or pH of the alkaline medium,exposure time, and temperature used in the treatment of the material tobe expanded.

ECM materials that can be processed to make expanded materials caninclude any of those disclosed herein or other suitable ECM's. Typicalsuch ECM materials will include a network of collagen fibrils havingnaturally-occurring intramolecular cross links and naturally-occurringintermolecular cross links. Upon expansion processing as describedherein, the naturally-occurring intramolecular cross links andnaturally-occurring intermolecular cross links can be retained in theprocessed collagenous matrix material sufficiently to maintain thecollagenous matrix material as an intact collagenous sheet material;however, collagen fibrils in the collagenous sheet material can bedenatured, and the collagenous sheet material can have analkaline-processed thickness that is greater than the thickness of thestarting material, for example at least 120% of the original thickness,or at least twice the original thickness.

Illustratively, the concentration of the alkaline substance fortreatment of the remodelable material can be in the range of about 0.5to about 2 M, with a concentration of about 1 M being more preferable.Additionally, the pH of the alkaline substance can in certainembodiments range from about 8 to about 14. In preferred aspects, thealkaline substance will have a pH of from about 10 to about 14, and mostpreferably of from about 12 to about 14.

In addition to concentration and pH, other factors such as temperatureand exposure time will contribute to the extent of expansion, asdiscussed above. In this respect, in certain variants, the exposure ofthe collagenous material to the alkaline substance is performed at atemperature of about 4 to about 45° C. In preferred embodiments, theexposure is performed at a temperature of about 25 to about 40° C., with37° C. being most preferred. Moreover, the exposure time can range fromat least about one minute up to about 5 hours or more. In someembodiments, the exposure time is about 1 to about 2 hours. In aparticularly preferred embodiment, the collagenous material is exposedto a 1 M solution of NaOH having a pH of 14 at a temperature of about37° C. for about 1.5 to 2 hours. Such treatment results in collagendenaturation and a substantial expansion of the remodelable material.Denaturation of the collagen matrix of the material can be observed as achange in the collagen packing characteristics of the material, forexample a substantial disruption of a tightly bound collagenous networkof the starting material. A non-expanded ECM or other collagenousmaterial can have a tightly bound collagenous network presenting asubstantially uniform, continuous surface when viewed by the naked eyeor under moderate magnification, e.g. 100× magnification. Conversely, anexpanded collagenous material can have a surface that is quitedifferent, in that the surface is not continuous but rather presentscollagen strands or bundles in many regions that are separated bysubstantial gaps in material between the strands or bundles when viewedunder the same magnification, e.g. about 100×. Consequently, an expandedcollagenous material typically appears more porous than a correspondingnon-expanded collagenous material. Moreover, in many instances, theexpanded collagenous material can be demonstrated as having increasedporosity, e.g. by measuring for an increased permeability to water orother fluid passage as compared to the non-treated starting material.The more foamy and porous structure of an expanded ECM or othercollagenous material can allow the material to be cast or otherwiseprepared into a variety of sponge or foam shapes for use in thepreparation of medical materials and devices. It can further allow forthe preparation of constructs that are highly compressible and whichexpand after compression. Such properties can be useful, for example,when the prepared medical graft material is to be compressed and loadedinto a deployment device (e.g. a lumen thereof) for delivery into apatient, and thereafter deployed to expand at the implant site.

After such alkaline treatments, the material can be isolated from thealkaline medium and processed for further use. Illustratively, thecollected material can be neutralized and/or rinsed with water to removethe alkalinity from the material, prior to further processing of thematerial to form a medical graft material of the invention.

Medical graft materials of the invention also can be used in conjunctionwith one or more secondary components to construct a variety of medicaldevices. In certain embodiments, the processed ECM material is affixedto an expandable member, such as a self-expanding or forcibly expandable(e.g. balloon-expandable) stent or a frame. Such devices of theinvention can be adapted for deployment within the cardiovascularsystem, including within an artery or vein. Certain devices are adaptedas vascular valves, for example for percutaneous implantation withinarteries, or within veins of the legs or feet to treat venousinsufficiency.

Prosthetic valve devices made with processed ECM materials of theinvention can be implanted into a bodily passage as frameless valvedevices or, as noted above, the ECM material can be attached to anexpandable frame. The ECM material can be used to form biocompatiblecoverings such as sleeves and/or to form leaflets or other valvestructures (see, e.g. WO 99/62431 and WO 01/19285). In one mode offorming a valve structure, the processed ECM material can be attached toa stent in a fashion whereby it forms one, two, or more leaflets, cusps,pockets or similar structures that resist flow in one direction relativeto another. In a specific application of such devices, such devicesconstructed as vascular valves are implanted to treat venousinsufficiencies in humans, for example occurring in the legs.

With reference now to FIG. 1, depicted is a sheet-form medical graft 10formed from a single layer of the inventive processed ECM material 11derived from tissue of a warm blooded vertebrate. FIG. 2 illustrates amedical graft device 20 formed from two layers of the inventiveprocessed ECM material 11. Sheet form medical graft devices as depictedin FIGS. 1 and 2 can be used in a variety of grafting applications asdescribed herein, including without limitation in wound care and softtissue support applications.

With reference now to FIGS. 3-5, depicted are various side views of aprosthetic valve device 31 of the invention. A processed ECM material ofthe invention is attached to a frame element 33 and provides twoleaflets 34 and 35 in a configuration for implantation in a patient. Inparticular, FIG. 3 provides a side view of prosthetic valve device 31taken in a direction parallel to the coapting upper edges 34 a and 35 aof leaflets 34 and 35. FIG. 4 provides a view of the device 31 depictedin FIG. 3 taken from the left side. FIG. 5 provides a view of the device31 depicted in FIG. 3 taken from the right side. Device 31 isparticularly well suited for vascular applications, such as implantationinto a vascular passage of a patient.

As can be seen from FIGS. 3-5, leaflets 34 and 35 include respectivefree edges 34 a and 35 a for coaptation with one another and respectivefixed edges 36 and 40 that will each be forced against the wall of avascular vessel upon implantation of device 31 in a path that partiallycircumscribes the vessel wall so as to each form a blood-capturingelement. In the device 31 illustrated, the path of leaflet edge contactwith the vessel wall includes substantial portions that extendessentially longitudinal along the vessel wall that connect to acup-forming portion that extends both longitudinally along the vesselwall and circumferentially around the vessel wall. In particular, thefixed edge 36 of leaflet 34 includes opposite longitudinally-extendingportions 37 and 38 each extending to an opposite side of a cup-formingportion 39. Correspondingly, the fixed edge 40 of leaflet 35 includesopposite longitudinally-extending portions 41 and 42 each extending toan opposite side of cup-forming portion 43.

The amount of contacting or coapting leaflet area can be expressed in anumber of different ways. The length of coaptation (e.g., LOC) in theoriginal configuration for implant is desirably at least about 2 mm andmay be as much as about 50 mm or more depending on the configuration ofthe valve prosthesis. In certain embodiments of the invention, thelength of coaptation can be within the range of about 5 to about 30 mm,more typically about 5 to about 15 mm, in the original configuration forimplant. The length of coaptation can represent a substantial percentageof the overall length of the valve prosthesis, for example, at leastabout 5%, or at least about 10%, of the overall length of theprosthesis. In certain embodiments, the length of coaptation of theleaflets represents 10% to 80% of the length of the overall device,typically about 30% to about 60%, and more typically about 35% to about55%.

In additional aspects, a long length of coaptation can be provided byorienting the outer leaflet edges substantially longitudinally along theframe in close proximity to one another over a significant distance.Thus, with reference to FIGS. 3-5 for purposes of illustration, outerleaflet edge portion 37 of leaflet 34 is configured to contact along thevessel wall in close proximity to outer leaflet edge portion 41 ofleaflet 35 over a significant distance, for example 2 to 50 mm,typically about 5 to about 30 mm, and more typically about 5 to about 15mm. The same would be true for the leaflet edge portions tracking alongthe opposite side of the vessel wall (e.g., edge portions 38 and 42,FIGS. 3-5). It is preferred that the leaflet edges remain in closeproximity over these distances, for example within about 5 mm, morepreferably within about 3 mm, and most preferably within about 1 mm. Itwill be understood that this close proximity may involve having leafletedges track closely with one another along the vessel wall, or may havethem being attached along essentially the same path (e.g., both along asingle strut of a frame) and thus exhibiting essentially no separationfrom one another as they pass along the vessel wall.

For the purpose of promoting a further understanding of aspects of thepresent invention, the following specific examples are provided. It willbe understood that these examples are illustrative and not limiting ofthe present invention.

Example 1

This example describes the preparation of an one processed ECM materialof the invention.

Porcine small intestines were received from a packing plant and weresectioned and split open to reveal their inner portions. After initialcleaning to remove the contents contained within the intestines, eachintestine was mechanically abraded on each side to remove mucosa andserosa layers and to isolate a primarily connective tissue layerincluding the submucosa for further processing. The submucosal layer wastreated in a 1:10 (wt:vol) 0.1% sodium dodecyl sulfate (SDS) solutionfor one hour at 37° C. followed by treatment in a 1:10 (wt:vol) 89 mMtris, borate, ethylene diamine tetraacetic acid (TBE) solution for onehour at 37° C. After these initial treatments, the submucosa was rinsedwith 1:10 (wt:vol) high purity water for 5 minutes at ambienttemperature. This rinsing step was repeated a second time beforetreating the submucosa in a 1:5 (wt:vol) 100% isopropyl alcohol (IPA)solution for 30 minutes at ambient temperature. This IPA treatment stepwas repeated a second time followed by rinsing the submucosa twice asdescribed above. The submucosa was then treated in a 1:10 (wt:vol) 0.2%PAA/5% specially denatured alcohol solution for two hours at ambienttemperature. Finally, the submucosa was rinsed in 1:10 (wt:vol) highpurity water for 5 minutes at ambient temperature. This rinsing step wasrepeated for a total of 4 rinses before testing the submucosa for SDSresidues. SDS content was measured by using a detergent detection kit(Chemetrics), which indicated that over 97% of the initial detergent wasremoved. The resulting submucosal tissue was used in the examples thatfollow.

Example 2

This example demonstrates an improved reduction in lipid content usingthe process described in Example 1 as compared to another process forpreparing a submucosal ECM material.

Ten lots of split porcine small intestine were obtained and split intotwo approximately equal groups. One group was processed as described inExample 1 of U.S. Pat. No. 6,206,931 (hereinafter referred to as the“control group”). Briefly, raw intestine was first treated withperacetic acid followed by mechanically abrading each side to isolatethe submucosal tissue layer and rinsing to remove chemical residues. Thesecond group was prepared as described above in Example 1 (hereinafterreferred to as the “test group”).

Each lot of material was used to make three 4-layer lyophilized sheetsby superimposing four of the submucosal layers (wet) and lyophilizingthe resultant stack. The 4-layer sheets were sterilized using alow-temperature ethylene oxide cycle. From each sheet, a 1 cm×1 cmsample was cut for lipid content analysis. Three samples were analyzedper group per lot (3 samples×10 lots) for a total of 30 samples for eachgroup. Each sample was weighed (initial weight) and then treated with asolution of 100% ethanol for 24 hours followed by a solution of acetonefor 24 hours to extract lipids. Samples were subsequently dried forapproximately 48 hours and weighed (final weight). Lipid content wascalculated by the initial weight minus the final weight divided by theinitial weight.

Distribution analysis was performed to determine which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the lipid content of the controlgroup best fit the log-normal distribution, which corresponds to anaverage lipid content of 8.06+/−5.59%. Distribution analysis indicatedthat the lipid content of the test group best fit the Weibulldistribution, which corresponds to an average lipid content of2.51+/−2.51%.

A p-value of 1.08×10⁻⁵ was achieved using an unpaired t-test on all ofthe samples. A p-value of 6.2×10⁻⁵ was achieved using a pairwise t-testto compare the lot matched control group results with the correspondingtest group results. Both p-values are less than 0.05, indicating thatthere is a statistically significant decrease in the lipid content ofthe test material as compared to the control material.

Example 3

This example demonstrates an improved reduction in IgA content using theprocess described in Example 1.

Ten lots of split porcine small intestine were obtained and split intotwo approximately equal groups. One group was processed as described inExample 1 of U.S. Pat. No. 6,206,931 (hereinafter referred to as the“control group”). Briefly, raw intestine was first treated withperacetic acid followed by abrading on each side to isolate a submucosaltissue layer and rinsing to remove chemical residues. The second groupwas prepared as described above in Example 1 (hereinafter referred to asthe “test group”).

Each lot of material was made into three, 4-layer lyophilized sheets bysuperimposing four of the submucosal layers (wet) and lyophilizing theresultant stack. The 4-layer sheets were sterilized using alow-temperature ethylene oxide cycle. From each sheet, a 1 cm×1 cmsample was cut for immunoglobulin A (IgA) content analysis. Threesamples were analyzed per group per lot (3 samples×10 lots) for a totalof 30 samples for each group. Each sample was weighed (initial weight),placed in a 1.5 mL centrifuge tube, and ground for 90 seconds in 400 μlof phosphate buffered saline (PBS). Samples were subsequentlycentrifuged, and the supernatant was isolated and diluted 1:5 withsterile PBS. These diluted samples were assayed for IgA content testingby ELISA using a kit from Bethyl Laboratories, Bethyl, Tex. IgA weightcontent was calculated by dividing the test group IgA content by theinitial weight of the control group.

Distribution analysis was performed to determine which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the control group IgA content bestfit the normal distribution, which corresponds to an average IgA contentof 50.4+/−27.7 μg/g. One of the test group samples tested at 1.54 μg/gwhile the all of the other test groups samples tested at 0 μg/g.Distribution analysis was therefore not performed for the test groupsince the distribution was essentially a single point.

A p-value of 1.7×10⁻¹³ was achieved using an unpaired t-test on all ofthe samples. A p-value of 1.88×10⁻⁴ was achieved using a pairwise t-testto compare the lot matched control group results with the correspondingtest group results. Both p-values are less than 0.05, indicating thatthere is a statistically significant decrease in the IgA content of thematerial of the test group as compared to the control group.

Example 4

This example demonstrates an improved reduction in the number of nucleiusing the process described in Example 1.

Ten lots of split porcine small intestine were obtained and split intotwo approximately equal groups. One group was processed as described inExample 1 of U.S. Pat. No. 6,206,931 (hereinafter referred to as the“control group”). Briefly, raw intestine was first treated withperacetic acid followed by abrading each side to isolate a submucosaltissue layer and rinsing to remove chemical residues. The second groupwas prepared as described above in Example 1 (hereinafter referred to asthe “test group”).

A sample from each lot in each group was cut and stained with Hoechst33258 for identifying nuclei. Three images of the nuclei in each samplewere obtained from random locations using an Olympus fluorescencemicroscope with an ultraviolet filter. Images were taken at a totalmagnification of 200× by the Spot Insight digital camera and acquiredthrough Spot RT computer software. These images represent a total areaof 0.263 mm². Three independent analyzers counted nuclei for each imageacquired. The number of nuclei per image was taken as the average ofthese 3 counts.

Distribution analysis was performed to determine which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the control group nuclei countsbest fit the Weibull distribution, which corresponds to average nucleiper field for the control group of 473+/−193 nuclei. Distributionanalysis indicated that the test group nuclei counts best fit the gammadistribution, which corresponds to average nuclei per field for the testgroup were 26.7+/−36.1 nuclei.

A p-value of 1.66×10⁻⁶ was achieved using a pairwise t-test to comparethe lot matched control group results with the corresponding test groupresults. The p-value is less than 0.05, indicating that there is astatistically significant decrease in the nuclei content of the testmaterial as compared to the control material.

Example 5

This example demonstrates that a processed ECM material as described inExample 1 retains native FGF-2.

Ten lots of raw porcine small intestine were obtained and processedaccording to Example 1. Each lot of material was made into three,4-layer lyophilized sheets as described in Example 2 above andsterilized using a low-temperature ethylene oxide cycle. From eachsheet, a 1 cm×1 cm sample was cut for FGF-2 content analysis. Threesamples were analyzed per lot (3 samples×10 lots) for a total of 30samples. Each sample was weighed (initial weight), placed into a 1.5 mLcentrifuge tube, and ground 3×30 seconds in 400 μl of phosphate bufferedsaline (PBS). Samples were subsequently centrifuged, and the supernatantwas isolated and diluted 1:5 with sterile PBS. These diluted sampleswere assayed in duplicate for FGF-2 content using R&D Systems FGF-2ELISA kits. FGF-2 weight content was calculated by dividing the FGF-2content as determined by ELISA by the initial weight of the sample.

Distribution analysis was performed to determine which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the FGF-2 content best fit thenormal distribution, which corresponds to a mean FGF-content value of25.0 ng/g+/−12.9 ng/g.

Example 6

This example demonstrates that a processed ECM material as described inExample 1 retains native hyaluronic acid (HA).

Ten lots of raw porcine small intestine were obtained and processedaccording to Example 1. Each lot of material was made into three,4-layer lyophilized sheets as described in Example 2 above andsterilized using a low-temperature ethylene oxide cycle. From eachsheet, a 1 cm×1 cm sample was cut for hyaluronic acid (HA) contentanalysis. Three samples were analyzed per lot (3 samples×10 lots) for atotal of 30 samples. Each sample was weighed (initial weight), placedinto a 1.5 mL centrifuge tube, and digested with 50 μl Proteinase K in450 μl of phosphate buffered saline (PBS) at 56° C. for 45 minutes.Samples were subsequently centrifuged and the supernatant was isolatedand diluted 1:40 with sterile PBS. These diluted samples were assayed induplicate for HA content by ELISA using a kit from Corgenics,Westminster, Colo. HA weight content was calculated by dividing the HAcontent as determined by ELISA by the initial weight of the sample.

Distribution analysis was performed to determined which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the test sample HA content best fitthe log-normal distribution, which corresponds to an average HA contentfor the test sample of 303+/−209 μg/g.

Example 7

This example demonstrates that a processed ECM material as described inExample 1 retains native sulfated glycosaminoglycans (sGAGs).

Ten lots of raw porcine small intestine were obtained and processedaccording to Example 1. Each lot of material was made into three,4-layer lyophilized sheets as in Example 2 above and sterilized using alow-temperature ethylene oxide cycle. From each sheet, a 1 cm×1 cmsample was cut for s(GAG) analysis. Three samples were analyzed per lot(3 samples×10 lots) for a total of 30 samples. Each sample was weighed(initial weight), placed into a 1.5 mL centrifuge tube, and digestedwith 50 μl Proteinase K in 450 μl of phosphate buffered saline (PBS) at56° C. for 45 minutes. All samples were vortexed for 5 seconds followedby the addition of 1.0 mL of Blyscan dye reagent. Absorbance readingswere taken with a spectrophotometer in triplicate from each sample at685 nm, and sGAG concentration was calculated from a heparin standardcurve. sGAG weight content was calculated by dividing the s(GAG) contentas determined by the absorbance readings by the total weight of sample.

Distribution analysis was performed to determine which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the sGAG content best fit thenormal distribution, which corresponds to a mean value of s(GAG) contentof 7588 μg/g+/−6505 μg/g.

Example 8

This example demonstrates the diaphragmatic burst force of the ECMmaterial that has been processed according to Example 1 as compared toanother process.

Ten lots of split porcine small intestine were obtained and split intotwo approximately equal groups. One group was processed as described inU.S. Pat. No. 6,206,931 (hereinafter referred to as the “controlgroup”). Briefly, raw intestine was first treated with peracetic acidfollowed by abrading on both sides to isolate a submucosal tissue layerand rinsing to remove chemical residues. The second group was preparedas described above in Example 1 (hereinafter referred to as the “testgroup”).

Each lot was made into three, 4-layer lyophilized sheets as described inExample 2 above or three, 8-layer vacuum pressed sheets by overlappingeight wetted submucosal layers and vacuum-pressing the construct. Thesesheets were sterilized using a low temperature ethylene oxide cycle.From each sheet, a 2.5 inch×2.5 inch sample was cut for diaphragmaticburst force testing. Three samples were analyzed per lot for the 4-layerlyophilized groups (3 samples×9 lots) for a total of 27 samples. Threesamples were analyzed per lot for the 8-layer vacuum pressed groups (3samples×10 lots) for a total of 30 samples. Each sample was rehydratedin phosphate buffered saline (PBS) for at least 15 minutes prior totesting. The diaphragmatic burst force was measured before breakingusing the Mullen's burst force tester.

Distribution analysis was performed to determined which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the control 4-layer lyophilizedburst force best fit the Weibull distribution, which corresponds to anaverage burst force of 359+/−98 kPa. Distribution analysis indicatedthat the test 4-layer lyophilized diaphragmatic burst force best fit thelog-normal distribution, which corresponds to an average burst force of378+/−79 kPa. The average difference in diaphragmatic burst forcebetween the two groups was 5.3%. A p-value of 0.458 was achieved usingan unpaired t-test on all of the samples. A p-value of 0.060 wasachieved using a paired t-test to compare the lot matched control groupresults with the corresponding test group results. Both of thesep-values are greater than 0.05, indicating that there was not astatistically significant decrease in the diaphragmatic burst force of a4-layer lyophilized material of the present invention as compared to a4-layer lyophilized material prepared by a currently used process.

With respect to the 8-layer vacuum pressed materials, distributionanalysis indicated that the control 8-layer vacuum pressed burst forcebest fit the log-normal distribution, which corresponds to an averageburst force of 915+/−234 kPa. Distribution analysis indicated that thetest 8-layer vacuum pressed burst force best fit the log-normaldistribution, which corresponds to an average burst force of 872+/−269kPa. The average difference in burst force between the two groups was4.8%. A p-value of 0.516 was achieved using an unpaired t-test on all ofthe samples. A p-value of 0.217 was achieved using a paired t-test tocompare the lot matched control group results with the correspondingtest group results. Both of these p-values are greater than 0.05,indicating that there was not a statistically significant decrease inthe burst force of the 8-layer vacuum-pressed test material as comparedto the 8-layer vacuum-pressed control material.

Example 9

This example demonstrates the suture retention strength of the ECMmaterial that has been processed according to Example 1 as compared toanother process for preparing a sterilized, isolated submucosa tissue.

Ten lots of split porcine small intestine were obtained and split intotwo approximately equal groups. One group was processed as described inU.S. Pat. No. 6,206,931 (hereinafter referred to as the “controlgroup”). Briefly, raw intestine was first treated with peracetic acidfollowed by abrading both sides to isolate a submucosal tissue layer andrinsing to remove chemical residues. The second group was prepared asdescribed above in Example 1 (hereinafter referred to as the “testgroup”).

Each lot was made into three, 4-layer lyophilized sheets or three,8-layer vacuum pressed sheets as described in Example 8. These sheetswere sterilized using a low temperature ethylene oxide cycle. From eachsheet, a 1 cm×3 cm sample was cut for suture retention strength testing.Three samples were analyzed per group per lot (3 samples×10 lots) for atotal of 30 samples for each group. The 4-layer lyophilized material wascut in the two primary directions, longitudinal and transverse, whilethe 8-layer material was only cut in one direction since this materialhas no primary direction due to the orthogonal overlapping of thelayers. Each sample was rehydrated in phosphate buffered saline (PBS)for at least 15 minutes prior to testing. A 302/304 stainless steelwire, equal in size to the 5-0 sutures used clinically, was passedthrough one end of each test article, with a bite depth of 2 mm, and wasattached to the movable jaw of the tensile testing machine. The otherend of each test article was gripped in the stationary jaw of thetensile testing machine and the wire was pulled upward at a constantrate of 150 mm/min. The maximum force, extension at break, and failuremode were recorded for each sample. All of the test samples (180/180,100%) failed as a result of the steel wire pulling out of the SISmaterial. In all instances, the steel wire remained intact and the SISmaterial failed.

The results of the tests are as follows (mean+/−standard deviation):8-layer vacuum pressed control group=12.22+/−2.68 N, 8-layer vacuumpressed test group=12.77+/−1.81 N (p=0.3714); 4-layer lyophilizedcontrol group transverse=7.58+/−1.68 N, 4-layer lyophilized test grouptransverse=7.99+/−1.90 N (p=0.3801); 4-layer lyophilized control grouplongitudinal=6.19+/−1.46 N, 4-layer lyophilized test grouplongitudinal=6.14+/−1.41 N (p=0.8929). Goodness of fit tests fornormality on the data sets showed that none of the data sets had anysignificant departures from a normal distribution. Therefore, two samplet-tests (control versus test groups) were run on each group of data(8-layer vacuum pressed, 4-layer lyophilized transverse, and 4-layerlyophilized longitudinal). All p-values were greater than 0.3714,indicating that there was no statistically significant decrease insuture retention strength in the test materials as compared to thecontrol materials.

Example 10

This example demonstrates the tensile strength of the ECM material thathas been processed according to Example 1 as compared to another processfor preparing a sterilized, isolated submucosal tissue.

Ten lots of split porcine small intestine were obtained and split intotwo approximately equal groups. One group was processed as described inU.S. Pat. No. 6,206,931 (hereinafter referred to as the “controlgroup”). Briefly, raw intestine was first treated with peracetic acidfollowed by abrading on both sides to isolate a submucosal tissue layerand rinsing to remove chemical residues. The second group was preparedas described above in Example 1 (hereinafter referred to as the “testgroup”).

Each lot was made into three, 4-layer lyophilized sheets or three,8-layered vacuum pressed sheets as in Example 8. These sheets weresterilized using a low temperature ethylene oxide cycle. From eachsheet, a “dog bone” shaped sample was cut for tensile testing. Threesamples were analyzed per group per lot (3 samples×10 lots) for a totalof 30 samples for each group. The 4-layer lyophilized material was cutin the two primary directions, longitudinal and transverse, while the8-layer material was only cut in one direction. Each sample wasrehydrated in phosphate buffered saline (PBS) and tested for theultimate tensile force (UTF) before breaking using the Instron at adeformation rate of 100 mm/minute.

Distribution analysis was performed to determine which distribution(normal, log-normal, Weibull, or gamma) best fit each set of data.Distribution analysis indicated that the control 4-layer lyophilized inthe longitudinal direction UTF best fit the log-normal distribution,which corresponds to an average UTF of 3.72+/−1.05 lbs. Distributionanalysis indicated that the test material 4-layer lyophilized in thelongitudinal direction UTF best fit the normal distribution, whichcorresponds to an average UTF of 2.92+/−0.88 lbs. The average differencein UTF between these two groups was 21%. A p-value of 3.6×10⁻³ wasachieved using an unpaired t-test on all of the samples. A p-value of3.4×10⁻³ was achieved using a paired t-test to compare the lot matchedcontrol group results with the corresponding test group results. Both ofthese p-values are less than 0.05, indicating that there was astatistically significant decrease in UTF in the longitudinal directionof the 4-layer lyophilized test material as compared to the 4-layeredlyophilized control material.

Similarly, distribution analysis indicated that the control 4-layerlyophilized in the transverse direction UTF best fit the log-normaldistribution, which corresponds to an average UTF of 3.2+/−0.96 lbs.Distribution analysis indicated that the test material 4-layerlyophilized in the longitudinal direction UTF best fit the log-normaldistribution, which corresponds to an average UTF of 2.48+/−0.90 lbs.The average difference in UTF between these two groups was 22%. Ap-value of 7.1×10⁻³ was achieved using an unpaired t-test on all of thesamples. A p-value of 5.11×10⁻² was achieved using a paired t-test tocompare the control group results with the corresponding test groupresults. Both of these p-values are less than 0.05, indicating thatthere was a statistically significant decrease in UTF in the transversedirection of the 4-layer lyophilized test material as compared to the4-layered lyophilized control material.

With respect to the 8-layer material, distribution analysis indicatedthat the control 8-layer vacuum pressed UTF best fit the Weibulldistribution, which corresponds to an average UTF of 10.78+/−3.35 lbs.Distribution analysis indicated that the test material 8-layer vacuumpressed UTF best fit the normal distribution, which corresponds to anaverage UTF of 7.46+/−2.45 lbs. The average difference in UTF betweenthese two groups was 31%. A p-value of 6.4×10⁻⁵ was achieved using anunpaired t-test on all of the samples. A p-value 9.9×10⁻⁷ was achievedusing a paired t-test to compare the lot matched control group resultswith the corresponding test group results. Both of these p-values areless than 0.05, indicating that there was a statistically significantdecrease in UTF of the 8-layer vacuum-pressed test material as comparedto the 8-layered vacuum-pressed control material.

Example 11

This example demonstrates that the processed ECM material of Example 1exhibits angiogenic character.

A disc for implantation was formed of porcine small intestinal submucosa(SIS) processed in the following ways: Control lyophilized (prepared asdescribed in Example 1 of U.S. Pat. No. 6,206,931), control vacuumpressed (prepared as described in Example 1 of U.S. Pat. No. 6,206,931),test lyophilized (prepared as described in Example 1), test vacuumpressed (prepared as described in Example 1), and a wound care productPromogran® (PR). Control and test discs were sterilized with a lowtemperature ethylene oxide cycle prior to implantation. The PR productwas received sterile and processed aseptically. Each disc was implantedinto the subcutaneous dorsi of a mouse for 3 weeks. After 3 weeks, eachdisc was probed for capillary formation. Angiogenesis was measuredqualitatively using fluorescence microangiography, a method of imagingintact, functional microvasculature. Vessel capacity, as a measure ofangiogenesis, was determined using vascular perfusion of fluorescentmicrospheres, followed by fluorescence extraction from the implantsusing xylenes, and subsequent quantitation with a fluorescence platereader. Lastly, implants were examined histologically by thin sectioningand H&E staining to illustrate cellular ingrowth.

Fluorescence microangiography indicated that all discs supportedangiogenesis. The test lyophilized disc had similar results to thecontrol lyophilized disc. The test vacuum pressed disc performedsuperior to the control vacuum pressed disc. The PR disc hadsignificantly less vascular growth with only one area for the twosamples investigated having any ingrowth. Vessel capacity results forthe PR disc were significantly less than the control. There was nostatistical difference for any of the other discs tested. Histologyconfirmed the microangiography findings. The lyophilized control discand the lyophilized test disc had significant fibrovascular ingrowth.The vacuum pressed control disc had only minor penetration. The vacuumpressed test disc had more cellular ingrowth, minoring the results ofthe microangiography, but still had signs of unincorporated SIS near theedge likely due to the dense nature of this material. Very littlecellular ingrowth was evident in the PR disc, suggesting very littleevidence of functional remodeling.

Example 12

This example provides a comparison of the processed ECM of Example 1with another processed ECM when used in a rat abdominal wall repairmodel.

Ten lots of split porcine small intestine were obtained and split intotwo approximately equal groups. One group was processed as described inExample 2 of U.S. Pat. No. 6,206,931 (hereinafter referred to as the“control group”). Briefly, raw intestine was first treated withperacetic acid followed by abrading on both sides to isolate asubmucosal tissue layer and rinsing. The second group was prepared asdescribed above in Example 1 (hereinafter referred to as the “testgroup”).

Each lot of material was made into a 4-layer vacuum pressed test implantor a 4-layer vacuum pressed control implant. A 2 cm×2 cm defect wascreated in the rat abdominal fascia and each implant was implanted toprovide the repair. After two, four, or eight weeks, the rats weresacrificed and implants were removed. A dog-bone shaped piece was cutfrom each implant and tested for mechanical strength.

The test implants were compared against their control counterparts ateach time point for mechanical strength at failure. In addition, atexplant, any complications were noted. Complications tallied includedinfection, abdominal adhesion formation, and seroma formation. At eachtime point, the test implant had similar UTF to failure compared to thecorresponding control implant. The test implant elicited less negativereaction from the host. Specifically, only one seroma was noted in thetest material at all points, while 5 seromas were seen in the controlimplant. Lastly, the histology indicated that the test implant remodeledas well as, if not better than, the control implant.

Example 13

This example describes the preparation of another processed ECM materialof the invention.

Porcine small intestines were received from a packing plant and weresectioned and split open to reveal their inner portions. After initialcleaning to remove the contents contained within the intestines, eachintestine was mechanically abraded on each side to remove mucosa andserosa layers and to isolate a primarily connective tissue layerincluding the submucosa for further processing. The submucosal layer wastreated in a 1:5 (wt:vol) 99% isopropyl alcohol (IPA) solution for 30minutes at ambient temperature. This IPA treatment step was repeated asecond time followed by rinsing the submucosa twice with tap water. Thesubmucosa was then treated in a 1:10 (wt:vol) 0.1% sodium dodecylsulfate (SDS) heated solution for one hour at 37° C. followed bytreatment in a 1:10 (wt:vol) 89 mM tris, borate, ethylene diaminetetraacetic acid (TBE) heated solution for one hour at 37° C. Thetreated submucosa was then rinsed twice with_tap water. After rinsing,the submucosa was treated in a 1:10 (wt:vol) 0.2% PAA/5% speciallydenatured alcohol solution for two hours at ambient temperature. Afterthese treatment steps, the submucosa was rinsed with 1:10 (wt:vol) highpurity water for 5 minutes at ambient temperature. This rinsing step wasrepeated for a total of 4 rinses before testing and release. The rinsewater was tested for pH, conductivity and PAA content. A sample ofsubmucosa was tested for bioburden after disinfection as well as forphysical attributes.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context. In addition, all publications cited herein areindicative of the abilities of those of ordinary skill in the art andare hereby incorporated by reference in their entirety as ifindividually incorporated by reference and fully set forth.

1.-36. (canceled)
 37. A method for preparing a medical graft material,comprising: providing a starting extracellular matrix (ECM) materialcontaining native IgA and native FGF-2; and treating said starting ECMmaterial effectively to retain the native FGF-2 at a level of at leastabout 10 ng/g while decreasing the native IgA content to a level lessthan 20 μg/g, wherein said treating comprises contacting the startingextracellular matrix material with an aqueous detergent solution. 38.The method of claim 37, wherein the method further comprises rinsing theECM material after said treating step so as to remove at least 90% ofthe detergent residues from the material. 39.-50. (canceled)
 51. Themethod of claim 37, wherein said detergent solution has a detergentconcentration of about 0.05% to about 1%.
 52. The method of claim 37,wherein said detergent solution has a detergent concentration of about0.05% to about 0.3%.
 53. The method of claim 37, wherein the detergentis sodium dodecyl sulfate.
 54. The method of claim 51, wherein thedetergent is sodium dodecyl sulfate.
 55. The method of claim 52, whereinthe detergent is sodium dodecyl sulfate.
 56. The method of claim 51,wherein said treating decreases the native IgA content to a level lessthan 15 μg/g.
 57. The method of claim 31, also comprising: prior to saidtreating, removing lipids from a precursor ECM material to said startingECM material by contact of the precursor ECM material with a polarorganic solvent.
 58. The method of claim 57, also comprising: after saidtreating, removing deoxyribonucleic acid from the ECM material bycontact with a basic aqueous medium in which deoxyribonucleic acid issoluble
 59. The method of claim 58, also comprising: after said removingdeoxyribonucleic acid, disinfecting the ECM material by contact with anoxidizing disinfectant.
 60. The method of claim 57, wherein the polarorganic solvent comprises isopropyl alcohol.
 61. The method of claim 58,wherein the polar organic solvent comprises isopropyl alcohol.
 62. Themethod of claim 59, wherein the polar organic solvent comprisesisopropyl alcohol.
 63. The method of claim 37, wherein the ECM materialcomprises renal capsule membrane, dermal collagen, dura mater,pericardium, fascia lata, serosa, peritoneum or basement membrane. 64.The method of claim 59, wherein, after said disinfecting, the ECMmaterial includes native FGF-2, native proteoglycans, and nativeglycosaminoglycans.
 65. The method of claim 51, wherein said detergentsolution is a heated detergent solution.
 66. The method of claim 54,wherein said detergent solution is a heated detergent solution.
 67. Amethod for preparing a medical graft material, comprising: treating anECM material with a liquid detergent medium so as to reduce native IgAin the ECM material to a level less than about 10 μg/g while retainingnative FGF-2 in the ECM material at a level of at least about 10 ng/gand while retaining native proteoglycans and glycosaminoglycans in theECM material.
 68. The method of claim 67, also comprising: treating theECM material with a liquid medium in which lipids are soluble so as toremove lipids from the ECM material; and treating the ECM material witha liquid medium in which deoxyribonucleic acid is soluble so as toremove deoxyribonucleic acid from the ECM.
 69. The method of claim 67,wherein said detergent medium has a detergent concentration of about0.05% to about 0.3%.